Breaks in the structure of DNA are a persistent stress on the integrity of the genome, and they pose a substantial risk of chromosomal rearrangement and genetic mutation that can challenge the well-being of an organism and promote the development of cancer. There are several cellular mechanisms that monitor the state of the genome and rapidly initiate repair mechanisms in response to DNA damage so that a healthy genome is passed on to the next generation. Poly(ADP-ribose) Polymerase-1, or PARP-1, is a primary responder to breaks in the structure of DNA. PARP-1 has a unique catalytic activity that synthesizes polymers of ADP-ribose as a posttranslational modification on target proteins, primarily on PARP-1 itself (automodification). Upon binding to DNA breaks, PARP-1 activity is turned on to modulate DNA damage repair pathways and thereby promote cell survival. In contrast, excessive DNA damage leads to an elevated level of PARP-1 activity that results in cell death. Regulation of PARP-1 activity is therefore a critical factor in determining the fate of a cell. Furthermore, inhibitors of PARP-1 have recently emerged as promising therapeutic agents for the treatment of cancer and inflammation. Despite a growing interest in PARP-1 inhibitors and the discovery of expanded roles for PARP-1 activity in DNA repair, transcriptional regulation, and apoptotic signaling, there are few insights into the mechanism of PARP-1 activity and regulation. The long-term objective of this research program is to establish at the molecular level the mechanisms that control PARP-1 activity. DNA damage is the most potent activator of PARP-1; therefore we have chosen to first focus on the mechanism of DNA-dependent activation of PARP-1. Using a combination of x-ray crystallography and biochemical analysis, the proposed research will advance our understanding of PARP-1 recognition of DNA damage, and PARP-1 interaction with chromatin (Specific Aim 1). These studies will provide the first views of PARP-1 bound to DNA and will therefore provide mechanistic insights that will significantly advance the PARP field of research, as well as having a more broad impact on the field of DNA repair and chromatin biology. The proposed work will demonstrate how multiple domains of PARP-1 collaborate to couple poly(ADP-ribose) synthesis to structure-specific DNA binding (Specific Aim 2). The detailed structural analysis of PARP-1 activity and regulation will improve current models of PARP-1 biological functions and potentially reveal novel strategies for specifically inhibiting PARP-1 activity. PUBLIC HEALTH RELEVANCE: Poly(ADP-ribose) Polymerase 1 (PARP-1) has important roles in DNA damage repair, regulation of gene expression, and cell death signaling. Inhibitors of PARP-1 have emerged as promising therapeutic agents for the treatment of cancer and inflammation. The studies in this proposal will advance our understanding of PARP-1 mechanism of action and regulation to improve current models of PARP-1 biological functions and to reveal novel strategies for inhibiting PARP-1.